Analysis of clinical outcomes and meiotic segregation modes following preimplantation genetic testing for structural rearrangements using aCGH/NGS in couples with balanced chromosome rearrangement

Abstract Purpose To retrospectively evaluate the effectiveness of PGT‐SR by array comparative genomic hybridization (aCGH) or next‐generation sequencing (NGS) in preventing recurrent miscarriages. Methods Thirty one couples with balanced translocation who underwent 68 PGT‐SR cycles between 2012 and 2020 were evaluated. A total of 242 blastocysts were biopsied for aCGH or NGS. The genetically transferable blastocysts were transferred in the subsequent frozen‐thawed single embryo transfer cycle. Results The genetically transferable blastocyst rate was 21.2% (51/241). Thirty five genetically transferable blastocysts were transferred into the uterine cavity. The clinical pregnancy rate was 57.1% (20/35), and the ongoing pregnancy rate was 100.0% (20/20). The incidence of interchromosomal effect (ICE) was influenced by ovarian stimulation protocol, female age, and carrier's gender, but dependent on the types of balanced translocation carriers. Furthermore, there was no significant difference in meiotic segregation modes in ovarian stimulation protocols and carrier's gender. Interestingly, the incidence of adjacent‐1 segregation in ≧40 years group increased significantly compared with <35 years group. Conclusions For the first time in Japan, we show the effectiveness of PGT‐SR using aCGH or NGS, which enables comprehensive analysis of chromosomes, in the prevention of recurrent miscarriages. Furthermore, our results may support better genetic counseling of balanced translocation carriers for PGT‐SR cycles.


| INTRODUC TI ON
Balanced structural chromosomal rearrangements such as reciprocal translocations (RecT), Robertsonian translocations (RobT), and inversions (Inv) are the most frequent chromosomal structural abnormalities. The phenotype of carriers with balanced translocations is normal, occurring in 0.2% of newborns and is found in 1%-5% of recurrent miscarriage couples. [1][2][3] However, they have a high risk of recurrent miscarriages or birth defects due to chromosomally abnormal embryos from the unbalanced gametes produced. [4][5][6][7] During meiosis of RecT, the translocated chromosomes and their normal homologs form quadrivalent chromosomes and cause adjacent-1, adjacent-2, 3:1, or 4:0 segregation. 8 Whereas, RobT has chromosomal rearrangements that result from the fusion of the entire long arms of two acrocentric chromosomes resulting in a trivalent chromosome that during meiosis results in chromosomal abnormality. 9 These abnormal gametes increase the risk of miscarriage, especially in firsttrimester abortions. In general, balanced translocation carriers have a high risk of recurrent miscarriage, approximately 50%-80% for RecT carriers and 50% for RobT. 3,10,11 Preimplantation genetic testing for structural rearrangements (PGT-SR) is an effective method of diagnosis for recurrent miscarriages in balanced translocation carriers. This method analyzes chromosomes using some of the cells of the preimplantation embryo and allows the selection of balanced/normal euploid embryos for embryo transfer. 12,13 Thus, it improves pregnancy outcomes in couples with balanced translocations, reducing the time to achieve a healthy live birth from 4-6 years to less than 4 months and decreasing the miscarriage rate to less than 15% in RecT carriers. 12,13 Previously, fluorescence in situ hybridization (FISH) was widely used for PGT-SR to distinguish balanced embryos from unbalanced ones. [14][15][16] FISH uses a probe that fluorescently labels a specific region of a chromosome to analyze the presence or absence of that particular region. Therefore, it is necessary to prepare several probes corresponding to chromosomal structural abnormalities for each translocation carrier. 17 In addition, many steps of the procedure depend on the technician's skill in preparing the specimen, which affects the diagnostic accuracy. 18 On the other hand, the clinical application of new technologies such as comprehensive chromosomal screening using array comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) has been shown to improve the clinical outcomes of PGT-SR. [19][20][21][22] These new methods require whole genome amplification (WGA).
They improve diagnostic accuracy because many processes are mechanized. Therefore, these have been now commonly used in PGT-SR chromosome analysis and recently it has been in use in Japan too. However, in Japan, there are only a few reports of the analysis by PGT-SR using these methods and clinical results are still unknown. 23 The chromosomal segregation on the spindle alignment in first meiosis is critical. In balanced translocation carriers, the chromosomes involved in the rearrangement would have a detrimental effect on the segregation of the structurally normal chromosomes. This is defined as the interchromosomal effect (ICE). 24 Since comprehensive chromosomal screening in PGT-SR is done now by aCGH or NGS, ICE is in focus. So far, translocation carriers with the acrocentric chromosome or telomere region have been reported to have a high chromosomal abnormality rate. 25 RobT carriers are shown to have more impact on ICE than RecT carriers, albeit in some contradictory reports. [26][27][28] However, it is still unclear how ovarian stimulation, female age at oocyte retrieval, carrier gender, and ICE impact chromosomal abnormalities during meiosis in translocation carriers.
This study aims to evaluate the efficacy of PGT-SR using aCGH or NGS in recurrent miscarriage prevention. In addition, we assessed the effects of female age, carrier gender, ovarian stimulation, etc., on chromosomal segregation during rearrangement in first meiosis.

| Study population
In this retrospective analysis, PGT-SR results were reviewed for 68 oocyte retrieval cycles of 31 couples from February 2012 to April 2020. Clinical indications for PGT-SR were reciprocal translocation in 26 couples, Robertsonian translocation in 4 couples, and pericentric inversion in 1 couple, with a previous clinical history of recurrent miscarriages in natural conception or IVF pregnancy (Table S1).
All patients underwent a systematic examination, including hysterosalpingography, diagnostic tests for antiphospholipid syndrome (APS), including screening for lupus anticoagulant by activated partial thromboplastin time (aPTT) and dilute Russell's viper venom time and (βeta2 glycoprotein I-dependent) anticardiolipin antibody, and blood tests for hypothyroidism and diabetes mellitus, before a subsequent pregnancy. The results of these diagnoses are shown in Table S1.

| Intracytoplasmic sperm injection, blastocyst culture, and biopsy
After oocyte retrieval, intracytoplasmic sperm injection (ICSI) was performed on metaphase II oocytes in all cases. 16-20 h after ICSI, those with 2 pronuclei were considered as normally fertilized oocytes and cultured to blastocysts. The embryos were cultured in sequential media (SAGE 1-step; CooperSurgical) at 37°C under 6.0% CO2, 5.0% O2 and 89.0% N2. Blastocysts were graded according to the Gardner blastocyst morphological scoring system. 29 Blastocysts with ≧3BB grades (3, 4, 5, 6, AA, AB, BA, and BB) were defined as good-quality blastocysts and those with C grade were defined as poor-quality blastocysts. On day 5 or 6 of the culture, the zona pellucida of the blastocysts was opened using a laser system (ZIROS-tk laser system; Hamilton Thorne Biosciences),

| Whole genome amplification, aCGH, and NGS
According to the manufacturer's protocol, the biopsied TE cells were  The establishment of pregnancy was determined at around 2 weeks after embryo transfer by the blood hCG concentration of ≧100 mIU/ml. Clinical pregnancy was determined at around 3 weeks after embryo transfer by detecting a single intrauterine gestational sac by transvaginal ultrasonography. It was considered as an ongoing pregnancy when no miscarriage was observed by 24 weeks.

| Statistics
Data are expressed as the mean ± standard deviation (SD).
Comparison of means was conducted by one-way analysis of variance (ANOVA) using Tukey's multiple range test. Comparisons of proportions were evaluated using the chi-square exact tests.
Statistical analyses were performed using StatView version 5.0 (SAS Institute). p < 0.05 was considered statistically significant. Table 1 summarizes the results of all PGT-SR cycles. The average age of females and males at oocyte retrieval was 37.0 and 39.2 years.

| Clinical outcomes of all PGT-SR cycles
Nine hundred twenty one921 cumulus-oocyte complexes (COCs) were retrieved, and ICSI was performed on 727 MII oocytes. Of these, 81.3% (591/727) confirmed normal fertilization having 2 pronuclei (2PN) and 62.6% (370/591) developed into blastocysts. A total of 242 blastocysts were biopsied and 16 of them were re-biopsied as the DNA amplification by WGA was unsuccessful. Despite re-biopsy, DNA amplification could not be confirmed in one of the blastocysts.
Of the 241 WGA successful blastocysts, the proportion of genetically transferable blastocysts (i.e., euploid, balanced, or mosaic; mosaic was defined as from 30% or more to less than 70% aneuploidy) was 21.2% (51/241). Thirty five genetically transferable blastocysts were transferred into the uterine cavity. The clinical pregnancy rate was 57.1% (20/35) and the ongoing pregnancy rate was 100.0% (20/20). In addition, the ongoing pregnancy rate per patient who underwent PGT-SR was 57.1% (16/28). Table 2 shows a comparison of the characteristics and clinical results of couples who were stimulated by GnRH agonist, GnRH antagonist, and mild stimulation protocols. There was no significant difference in the average age of females and males at oocyte retrieval between In the results of oocyte retrieval and embryo culture, there was no difference in the mean number of retrieved COCs between GnRH agonist (17.7) and GnRH antagonist (18.3), but it was significantly decreased in the mild stimulation group (4.3). In addition, there was no difference in the normal fertilization rate, the blastocyst formation rate, and morphologically good blastocyst rate among the protocols. There were no significant differences between GnRH agonist (68.0%) and GnRH antagonist (68.6%) in the biopsied blastocyst rates (blastocysts that could be biopsied), but they were significantly decreased in the mild stimulation group (44.4%). Moreover, there was no difference in the proportion of genetically transferable blastocysts between GnRH agonist (22.3%) and GnRH antagonist (22.9%), but it was significantly decreased in the mild stimulation group (5.0%).

| Comparison of stimulation protocol
In the results of embryo transfer, there were no significant differences in the female age at embryo transfer, morphologically good blastocyst rate, clinical pregnancy rate, and ongoing pregnancy rate between GnRH agonist and GnRH antagonist. On the other hand, as there was only one cycle for mild stimulation protocol, it was not possible to compare by statistical analysis for embryo transfer.
Nevertheless, one genetically transferable blastocyst was obtained, leading to ongoing pregnancies post frozen-thaw blastocyst transfer. Table 3 shows a comparison of the characteristics and clinical outcomes by the female age at oocyte retrieval, which was divided into Furthermore, there were no significant differences in the morphologically good blastocyst rate and the biopsied blastocyst rate among the groups. However, there was no difference in the rate of genetically transferable blastocysts between<35 years (17.8%) and the 35-39 years groups (29.7%), but no genetically transferable blastocysts were obtained in ≧40 years group.

| Comparison of female age at oocyte retrieval
In the results of embryo transfer, although there was a difference in maternal age at embryo transfer between <35 years (33.1) and 35-39 years groups (37.8), there was no difference in morphologically good blastocyst rate, clinical pregnancy rate, and ongoing pregnancy rate.    100.0 (9/9) 100.0 (11/11) -Note: Values for each parameter are presented as the mean ± standard deviation (SD).

| Chromosomal abnormality analysis for structural rearrangement in blastocysts of RecT carriers
Different letters indicate significant differences (p < 0.05).

TA B L E 3
The characteristics and clinical outcomes by female age at oocyte retrieval blastocysts, the incidence of adjacent-1 segregation was 58.9%, adjacent-2 segregation was 25.8%, and 3:1 segregation was 15.3%.
First, we evaluated the effect of the ovarian stimulation protocols on the meiotic segregation modes and there was no significant difference between the three stimulation cycles. Next, the influence of female age at oocyte retrievals on the meiotic segregation modes was evaluated. There was no significant difference in the incidence of adjacent-2 segregation and 3:1 segregation among the 3 female age groups at oocyte retrievals. Interestingly, there was a significant difference in the incidence of adjacent-1 segregation between <35 years (52.2%) and ≧40 years group (76.2%), but there was no difference in 35-39 years groups (57.9%). Then, we evaluated the effect of the carrier's gender on the meiotic segregation modes and found no significant differences.

| DISCUSS ION
In the present study, to evaluate the efficacy of PGT-SR by aCGH or NGS for preventing recurrent miscarriages in balanced translocation carriers, we did a retrospective analysis of the chromosomal status of the embryos that underwent PGT-SR. Subsequently, we analyzed the clinical outcomes of the frozen-thawed embryo transfer cycles of those embryos ( Note: Values for each parameter are presented as the mean ± standard deviation (SD).
Therefore, it is probable that there was no significant improvement in the live birth rates in this study.
Regarding the factors that influence chromosomal abnormalities in blastocysts, we analyzed ovarian stimulation, female age, carrier gender, and types of balanced translocations. In the present study, the optimal ovarian stimulation protocol for producing good quality blastocysts suitable for PGT-SR could not be clearly shown. There was no significant difference in the number of genetically transferable blastocysts between the GnRH agonist and GnRH antagonist protocols (Table 2). Moreover, there was no significant difference between these two groups regarding ICE in which translocated chromosomes affect the meiosis segregation of nontranslocated chromosomes (Table 6). Therefore, it is thought that differences in ovarian stimulation protocols might not affect the incident rate of aneuploidy in the blastocysts. It has also been shown that there is an increase in the incident rate of aneuploidy in the blastocysts when the total Gn dosage is low. 35 However, in the present study, there was no significant difference in the initiation and total dosage of Gn between these two ovarian stimulation protocols (Table S2).
Therefore, there was no difference in the genetically transferable blastocyst rates between them.
On the contrary, the genetically transferable blastocyst rates were significantly reduced in the mild stimulation with a low Gn dosage (Tables 2 and S2), which is considered to be due to the increase in female age at oocyte retrievals. 36 Regarding the factors that influence chromosomal abnormalities, in the female age at oocyte retrieval, genetically transferable blastocysts could not be obtained in ≧40 years group compared with the younger age groups (<35 years and 35-39 years; Table 3). Although the unbalanced blastocyst rate was lower in the≧40 years group, the sum of unbalanced blastocyst and unbalanced + aneuploid blastocyst did not differ between the three groups (60.0%, 58.5%, and 63.6; Table 6), as others have reported. 28,37 However, the aneuploid blastocyst and total ICE rate in ≧40 years group were higher than in the younger age groups. From the above, the results suggest that female age is a significant factor affecting chromosome abnormalities.
Carrier gender may affect chromosome segregation during meiosis, with different segregation patterns reported in females and males. 30,38,39 In the present study, the genetically transferable  Different letters indicate significant differences (p < 0.05).

TA B L E 5
The characteristics and clinical outcomes by type of rearrangement blastocyst rates were about 20% for both female and male carriers (Table 4), in agreement with other reports. 30,40,41 Although the unbalanced blastocysts in male carriers were significantly fewer than in female carriers, there was no difference in the unbalanced blastocyst rates, including Unbalanced + Aneuploid blastocysts (Table 6). Though these results agreed with the previous studies, the aneuploidy of nontranslocated chromosomes did not match. 40,42 Previous reports have also shown that the carrier gender does not affect the aneuploidy in nontranslocated chromosomes. 30,38,39 In this study, the average female age at the oocyte retrieval (36.9) was higher than in previous studies (30 ~ 32). 39,42 Therefore, it was considered that the aneuploid blastocyst rate increased due to the increase in chromosomal abnormalities due to the increase in female age. However, aneuploidy of nontranslocated chromosomes was significantly increased in male carriers in the present study. One of the factors could be the low rate of morphologically good blastocyst formation in male carriers. Chromosomal aneuploidy is associated with blastocyst evaluation and morphologically poor blastocysts have been shown to have high rates of aneuploidy. 43,44 In the report of embryo observation using the time-lapse system, embryos with delayed development have shown a high aneuploidy rate. 45,46 Therefore, it was thought that the aneuploid blastocysts rate was Note: Different letters indicate significant differences (p < 0.05).
RecT carriers (Table 6), supporting the expected segregation pattern in RecT and RobT carriers. In addition, RecT carriers had more abnormalities in nontranslocated chromosomes and particularly, the proportion of Unbalanced + Aneuploid blastocysts was significantly higher (Table 6). Some reports focusing on ICE show that RecT carriers have a higher abnormality in nontranslocated chromosomes containing unbalanced chromosomes. 27,40 In addition, the incidence of ICE by the location of breakpoints on the RecT chromosomes has been reported to be high in short-arm translocations. 26 The difference in the location of breakpoints in RecT would affect the rearrangement and pairing of translocated chromosomes and normal homologous chromosomes compared with RobT.
Ovarian stimulation protocol, female age at oocyte retrieval and carrier's gender were the parameters for the analysis of the segregation modes of reciprocal translocation. (Table 7). We observed no difference in the segregation pattern in any of the ovarian stimulation protocols. In addition, there was no difference in the segregation pattern due to the carrier's gender, which corresponded with previous reports. 25 On the other hand, in the female age at oocyte retrieval, there was a significant increase in the incidence of adjacent-1 segregation in ≥40 years group compared with the 35 years group, but there was no difference was in the 35-39 years group. Moreover, we also compared whether the translocation chromosome contained an acrocentric chromosome (Table S3), the translocation breakpoint was on the short arm or the long arm (Table S4) and terminal breakpoints (Table S5) among the 3 female age groups. As a result, there were no differences in the acrocentric chromosomes, the chromosome arms with breakpoints and the terminal breakpoints, and no association was found with female age at oocyte retrieval. Adjacent-1 segregation separates chromosomes with nonhomologous centromeres and adjacent-2 segregation separates chromosomes with homologous centromeres during meiosis in a reciprocal translocation heterozygote. In meiosis, sister chromatids and their centromeres are initially bound by cohesin. In anaphase I, cohesin on the sister chromatid arms is degraded, but around the centromere is not. However, it has been reported that cohesin decreases with age. 48,49 Therefore, we suspected that as the binding of the centromere region could not be maintained by cohesin, the adjacent-1 segregation increased due to the separation of the sister chromatid.
Here, for the first time in Japan, we present the clinical results of PGT-SR by aCGH or NGS, which enables a comprehensive analysis of chromosomes. Notably, the transfer of normal/balanced blastocysts did not result in any miscarriages in this study. Therefore, it can be assumed that PGT-SR by aCGH/NGS is effective in selecting embryos with chromosomal normality thus preventing recurrent miscarriages. However, the genetically transferable embryos cannot be obtained in female translocation carriers of ≧40 years, and PGT-SR is considered to be few effective in leading to live birth. Therefore, it is considered that sufficient explanation is necessary for the implementation of PGT-SR in ≧40 years old. Furthermore, our results may help predict the segregation pattern by comparing various factors leading to better genetic counseling of balanced translocation carriers for PGT-SR cycles using blastocyst biopsy.

ACK N OWLED G M ENTS
We are grateful to OVUS Co., Ltd. and Genesis Genetics Asia Co., Ltd. for supporting the chromosome analysis of this study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest associated with this manuscript. All procedures followed were in accordance with the ethical standards of the concerned institutional and national committees for human experimentation and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained